VIII. METHODS FOR TYPING MYCOBACTERIA
349
tion of agar and Malachite green, as incorporated in L-J medium, reduced
the number of plaques. Also, surface active agents such as Tween-80 were
found to be detrimental.
As a result of information obtained from these tests, a new medium for
bacteriophage studies on the mycobacteria was formulated (Redmond and
Ward, 1966). This is the RVA, RVB medium that has been tested in several
laboratories in a number of different countries. It has been recommended
by the International Committee on Phage Typing of Mycobacteria associated with the various laboratories involved in the phage typing studies.
Only slight differences other than the addition of agar exist between RVA
and RVB media; however, the formulae and procedure for making each
are given.
A. RVA-17 agar medium
It has been found necessary to follow certain definite procedures for
combining some of the chemical substances in order to prevent undesirable
reactions. For best results these procedures should be followed in making
the medium.
RVA- 17
Solution A
NazHP04 (anhydrous)
KHzP04
NaCl
NH4C1
Sodium pyruvate
Nutrient broth (Difco)
Proteose peptone No. 3 (Difco)
Casein hydrolysate (enzymatic)
Glycerol
ZnS04
C u S 0 4 . 5 H ~ O
Distilled water
Autoclave at 15 lb pressure for 20 min.
1.2 g
0.55 g
2.5 g
0.5 g
0.75 g
2.5 g
8.0 ml
0.10 mg
0.10 mg
360 ml
2.0 g
2.0 g
Solution B
Glucose (anhydrous)
5.0 g
Distilled water
50 ml
Autoclave at 15 lb pressure for 15 min.
Solution C
MgS04.7Hz0 (1 M stock solution) (page 352)
CaC12 (1 M stock solution) (page 352)
1 ml
1 ml